Highly fluorescent carboxyl-containing indocyanine dyes are useful as labeling reagents for biological investigations. Functional groups on the dyes permit covalent bonding to biomaterials and/or nonbiological materials for purposes of fluorescence detection of the labeled material and, depending upon the functional groups, such covalent bonding can be selective. See Alan S. Waggoner, "Fluorescent Probes for Analysis of Cell Structure, Function, and Health by Flow and Imaging Cytometry"; Applications of Fluorescence in the Biomedical Sciences, pages 3-28, 1986 Alan R. Liss, Inc. Depending upon the material, dyes containing selected functional groups will or will not bind to the material. The incorporation of carboxylic acid groups into the basic cyanine structure is expected to permit fluorescent labeling through the use of derived active esters. Thus, it is of interest to synthesize compounds containing carboxylic acid groups that will provide for the construction of the carboxyl-containing indocyanine dyes.
(2,3,3-Trimethyl-3-H-indol-5-yl)-acetic acid (3) is expected to possess exceptional advantages as the precursor of a family of dyes of cyanine and related types intended for use as covalently attached fluorescent labels for biological research. Disclosed herein is a convenient synthesis of 3 and its conversion to derivatives used to prepare carboxy cyanines.
Because the hydrazone 2 deteriorated rapidly during storage, attention was directed to "one pot" versions of the Fischer synthesis. However, procedures in the prior art proceeded slowly and gave poor yields of 3. See B. Robinson, "The Fischer Indole Synthesis," John Wiley & Sons, Inc., New York, N.Y. (1985) p. 640. It was found that, in acetic acid, hydrazone formation is nearly complete within 30 minutes in the presence of acetate ion. Indolization to 3 (UV max 260 nm) is essentially complete after 30 minutes at reflux. The generation of small amounts of a purple-red by-product often accompanied indolization, generally to a greater extent when sodium acetate had been added than when it had not. Yields of recrystallized product of satisfactory purity were 60% based on the arylhydrazine hydrochloride 1. ##STR1##
Compound 3 is a fluorescent substance; its solutions produce spots with a blue-white fluorescence on thin-layer plates when irradiated at 360 nm. Reaction with ethyl iodide yielded the ethiodide 4, which was converted to the highly fluorescent carbocyanine 5 (red fluorescence) by treatment with triethyl orthoformate in refluxing pyridine. See F. M. Hamer, J. Chem. Soc., 2796 (1927) for the examples of this method. Reaction of 3 with 1,4-butanesultone 6) afforded 7, a type of quaternary salt useful as an intermediate for water-soluble cyanine dyes. See United Kingdom Patent No. 742,112 (1955), Agfa A. G. fur Photofabriken; Chemical Abstracts, 50, 111496 (1956).